1. Field of the Invention
The invention relates to the field of recombinant DNA technology, more in particular to the field of gene therapy. In particular the invention relates to novel methods of delivering DNA to target cells and the subsequent integration of that DNA into the target cell genome.
2. State of the Art
In the field of gene therapy, many different methods have been developed to introduce new genetic information into target cells. Currently, the most efficient means of introducing DNA into target cells is by employing modified viruses, so-called recombinant viral vectors. The most frequently used viral vector systems are based on retroviruses, adenoviruses, herpes viruses or the adeno-associated viruses (AAV). All systems have their specific advantages and disadvantages. Some of the vector systems possess the capacity to integrate their DNA into the host cell genome, whereas others do not. From some vector systems the viral genes can be completely removed from the vector while in other systems this is not yet possible. Some vector systems have very good in vivo delivery properties, while others do not. Some vector types are very easy to produce in large amounts, while others are very difficult to produce.
The present invention combines functional components of two vector systems, thereby combining the favorable properties of both vector systems. The present invention was made during research involving adenovirus and adeno-associated virus. The invention typically provides DNA having a packaging signal which allows it to be encapsidated into virus particles of viruses which allow for encapsidation of large nucleic acids, such as adenovirus particles, which DNA (at least a part thereof) has the capacity to integrate into the host cell genome. The invention also provides for methods to ensure the absence of harmful viral genes from the encapsidated DNA. Absence of viral genes from the vector is the best way to avoid expression of viral gene products in target cells and thus the best way to circumvent immune responses to viral gene products expressed by transduced target cells.
The present invention can convey the above properties onto adenovirus vectors but also to other viruses, such as herpes or polyomaviruses.
The invention will, however, be explained in more detail based on adenovirus and adeno-associated virus vectors. Currently, adenovirus vectors attract a lot of attention and it is expected that the first registered gene therapy medicine will carry the foreign gene into the diseased cells of the patient through adenovirus vector mediated gene transfer. An important problem regarding adenovirus vectors is that they do not integrate into the host cell genome. In rapidly dividing tissue, such as the hemopoietic system, the vector is rapidly lost. Another problem with the current generation of adenovirus vectors is that they are immunogenic. In vivo, vector infected cells are cleared from the body by a potent immune reaction involving both a cellular and a humoral immune component.
For the purpose of gene therapy, adenoviruses carrying deletions have been proposed as suitable vehicles. Gene-transfer vectors derived from adenoviruses (so-called adenoviral vectors) have a number of features that make them particularly useful for gene transfer for such purposes. E.g. the biology of the adenoviruses is characterized in detail, the adenovirus is not associated with severe human pathology, the virus is extremely efficient in introducing its DNA into the host cell, the virus can infect a wide variety of cells and has a broad host-range, the virus can be produced in large quantities with relative ease, and the virus can be rendered replication defective by functional deletion of the early-region 1 (E1) of the viral genome.
During the productive infection cycle, the viral genes are expressed in two phases: the early phase, which is the period up to viral DNA replication, and the late phase, which coincides with the initiation of viral DNA replication. During the early phase only the early gene products, encoded by regions E1, E2, E3 and E4, are expressed, which carry out a number of functions that prepare the cell for synthesis of viral structural proteins (Berk, 1986). During the late phase the late viral gene products are expressed and the early gene products and host cell DNA and protein synthesis are shut off. Consequently, the cell becomes dedicated to the production of viral DNA and of viral structural proteins (Tooze, 1981).
The E1 region of adenovirus is the first region of adenovirus expressed after infection of the target cell. This region consists of two transcriptional units, the E1A and E1B genes, which both are required for oncogenic transformation of primary (embryonal) rodent cultures. The main functions of the E1A gene products are i) to induce quiescent cells to enter the cell cycle and resume cellular DNA synthesis, and ii) to transcriptionally activate the E1B gene and the other early regions (E2, E3, E4). Transfection of primary cells with the E1A gene alone can induce unlimited proliferation (immortalization), but does not result in complete transformation. However, expression of E1A in most cases results in induction of programmed cell death (apoptosis), and only occasionally immortalization is obtained (Jochemsen et al., 1987). Co-expression of the E1B gene is required to prevent induction of apoptosis and for complete morphological transformation to occur. In established immortal cell lines, high level expression of E1A can cause complete transformation in the absence of E1B (Roberts et al., 1985). The E1B encoded proteins assist E1A in redirecting the cellular functions to allow viral replication. The E1B 55 kD and E4 33 kD proteins, which form a complex that is essentially localized in the nucleus, function in inhibiting the synthesis of host proteins and in facilitating the expression of viral genes. Their main influence is to establish selective transport of viral mRNAs from the nucleus to the cytoplasm, concomitantly with the onset of the late phase of infection. The E1B 21 kD protein is important for correct temporal control of the productive infection cycle, thereby preventing premature death of the host cell before the virus life cycle has been completed. Mutant viruses incapable of expressing the E1B 21 kD gene-product exhibit a shortened infection cycle that is accompanied by excessive degradation of host cell chromosomal DNA (deg-phenotype) and in an enhanced cytopathic effect (cyt-phenotype) (Telling et al., 1994). The deg and cyt phenotypes are suppressed when, in addition, the E1A gene is mutated, indicating that these phenotypes are a function of E1A (White et al., 1988). Furthermore, the E1B 21 kD protein slows down the rate by which E1 A switches on the other viral genes. It is not yet known through which mechanism(s) the E1B 21 kD protein quenches these E1A dependent functions.
Vectors derived from human adenoviruses, in which at least the E1 region has been deleted and replaced by a gene-of-interest, have been used extensively for gene therapy experiments in the pre-clinical and clinical phase.
The adenovirus genome is a linear double-stranded DNA molecule of approximately 36,000 base pairs with the 55 kD terminal protein covalently bound to the 5xe2x80x2 terminus of each strand. The Ad DNA contains identical Inverted Terminal Repeats (TR) of about 100 base pairs with the exact length depending on the serotype. The viral origins of replication are within the TRs exactly at the genome ends. DNA synthesis occurs in two stages. First, the replication proceeds by strand displacement, generating a daughter duplex molecule and a parental displaced strand. The displaced strand can form a so-called xe2x80x9cpanhandlexe2x80x9d intermediate, which allows replication initiation and generation of a daughter duplex molecule. Alternatively, replication may go from both ends of the genome simultaneously, obliterating the requirement to form the panhandle structure. The replication is summarized in FIG. 1 adapted from (Lechner and Jr., 1977).
As stated before, all adenovirus vectors currently used in gene therapy have a deletion in the E1 region, where novel genetic information can be introduced. The E1 deletion renders the recombinant virus replication (Stratford-Perricaudet and Perricaudet, 1991). We have demonstrated that recombinant adenoviruses are able to efficiently transfer recombinant genes to the rat liver and airway epithelium of rhesus monkeys (Bout et al., 1994a; Bout et al., 1994b). In addition, we (Vincent et al., 1996a; Vincent et al., 1996b; Vincent et al., 1996c) and others (see e.g. (Haddada et al., 1993)) have observed a very efficient in vivo adenovirus mediated gene transfer to a variety of tumor cells in vitro and to solid tumors in animal models (lung tumors, glioma) and human xenografts in immunodeficient mice (lung) in vivo (reviewed (Blaese et al., 1995)).
In contrast to, for instance, retroviruses, adenoviruses a) are able to infect non-dividing cells and b) are able to efficiently transfer recombinant genes in vivo (Brody and Crystal, 1994). Those features make adenoviruses attractive candidates for in vivo gene transfer of, for instance, suicide or cytokine genes into tumor cells.
However, a problem associated with the current recombinant adenovirus vectors is that they do not integrate into the host cell genome. Due to this fact the vector is rapidly lost in dividing tissue. Recently it was demonstrated that integration of adenovirus vectors can be achieved in fertilized oocytes by using extreme multiplicities of infection (Tsukui et al., 1996). In somatic cell gene therapy this is an extremely undesired feature. Efficient integration of adenovirus vectors has also been observed in vitro in cells in which DNA damage was introduced by ionizing irradiation (Zeng et al., 1997). This is a very harsh treatment and not favored in gene therapy protocols.
One of the additional problems associated with the use of recombinant adenovirus vectors is the host-defense reaction against treatment with adenovirus.
Briefly, recombinant adenoviruses are deleted for the E1 region (see above). The adenovirus E1 products trigger the transcription of the other early genes (E2-E4), which consequently activates expression of the late virus genes. Therefore, it was generally thought that E1 deleted vectors would not express any other adenovirus genes. However, recently it has been demonstrate that some cell types are able to express adenovirus genes in the absence of E1 sequences. This indicates that some cell types possess the machinery to drive transcription of adenovirus genes. In particular, it was demonstrated that such cells synthesize E2A and late adenovirus proteins.
In a gene therapy setting, this means that transfer of the therapeutic recombinant gene to somatic cells not only results in expression of the therapeutic protein but also in the synthesis of viral proteins. Cells that express adenoviral proteins are recognized and killed by Cytotoxic T Lymphocytes, which thus a) eradicates the transduced cells and b) causes inflammations (Bout et al., 1994a; Engelhardt et al., 1993; Simon et al., 1993). As this adverse reaction is hampering gene therapy, several solutions to this problem have been suggested, such as a) using immunosuppressive agents after treatment; b) retainment of the adenovirus E3 region in the recombinant vector (see patent application EP 95 20 2213) and c) using ts mutants of human adenovirus, which have a point mutation in the E2A region. However, these strategies to circumvent the immune response have their limitations.
The use of ts mutant recombinant adenovirus diminishes to some extent the immune response, but was less effective in preventing pathological responses in (Engelhardt et al., 1994a).
The E2A protein may induce an immune response by itself and it plays a pivotal role in the switch to the synthesis of late adenovirus proteins. Therefore, it is attractive to make recombinant adenoviruses which are mutated in the E2 region, rendering it temperature sensitive.
A major drawback of this system is the fact that, although the E2 protein is unstable at the non-permissive temperature, the immunogenic protein is being synthesized. In addition, it is to be expected that the unstable protein does activate late gene expression, albeit to a low extent. ts125 mutant recombinant adenoviruses have been tested, and prolonged recombinant gene expression was reported (Engelhardt et al., 1994a; Engelhardt et al., 1994b; Yang et al., 1995; Yang et al., 1994). However, pathology in the lungs of cotton rats was still high (Engelhardt et al., 1994a), indicating that the use of ts mutants results in a partial improvement in recombinant adenovirus technology. An additional difficulty associated with the use of ts125 mutant adenoviruses is that a high frequency of reversion is observed. These revertants are either real revertants or second site mutations (Kruijer et al., 1983; Nicolas et al., 1981). Both types of revertants have an E2A protein that functions at normal temperature and have therefore similar toxicity as. the wild-type virus.
In adeno-associated virus vectors the entire protein coding domain can be replaced by foreign sequences. Adeno-associated virus vectors can integrate into the host cell genome (Kotin, 1994). The only AAV-sequences required in the vector are the inverted terminal repeat elements flanking the foreign DNA. Due to the integrating properties and the absence of viral genes AAV-vectors are very well suited for the permanent genetic modification of target cells in vivo. One drawback is, however, that they are very difficult to produce. Another drawback is the limited packaging size. Only molecules up to approximately 5 kb are efficiently packaged. Another drawback is that rAAV vectors arc delivered as single strand DNA molecules. In the target cell a second complementary strand has to be produced for expression to occur. This does not occur immediately after infection with rAAV. Second strand synthesis is indeed the rate limiting step for expression of the transgene (Ferrari et al., 1996).
The present invention provides methods and means to combine the integrating capacity of one virus with the large packaging and infection capacity of another virus, as well as the results of these methods and the use of these results. The present invention thus also provides methods to combine the favorable properties of adenovirus vectors with the favorable properties of AAV-vectors.
The present invention provides methods to completely remove all viral genes from the vector thus completely avoiding the cellular immune responses to viral gene products synthesized in the target cell. The only adenovirus sequences necessarily present in the encapsidated DNA are those comprising a functional packaging signal. In cis required sequences for multiplication of vector genomes in the virus producing cell are functional AAV-TR sequences at both ends of the DNA. This Ad/AAV chimeric molecule is replicated in the vector producing cell by the AAV-replication machinery. Packaging of the Ad/AAV chimeric molecules into adenovirus capsids is achieved following expression of the relevant adenovirus genes involved in packaging DNA into adenovirus capsids.
AAV is a non-pathogenic human parvovirus (reviewed in (Berns, 1990a; Berns, 1990b)). The virus replicates as a single strand DNA of approximately 4.6 kb. Both the plus and the minus strand are packaged and infectious. Efficient replication of AAV requires the co-infection of the cell by a helper virus such as Adenovirus or Herpes Simplex Virus. In the absence of a helper virus no substantial replication of AAV is observed. AAV is therefore also classified as a xe2x80x9cDependovirusxe2x80x9d. When no helper virus is present, the AAV genome can integrate into the host cell genome. The wild-type virus has a strong preference (70%) for an integration site on the long arm of chromosome 19 (19 q13.3) (Kotin et al., 1990; Samulski, 1993; Samulski et al., 1991). This site specificity is probably mediated by the AAV-rep proteins, more specifically by Rep78 and Rep68 (Weitzman et al., 1994). Following integration, the expression of the virus genes is not detectable. The integrated provirus replicates as a normal part of the host cell genome upon division of the transduced cell and ends up in both daughter cells. This stage of the virus life cycle is known as the latent stage. This latent stage is stable but can be interrupted by infection of the transduced cell by a helper virus. Following infection of the helpervirus, AAV is excised from the host cell genome and starts to replicate. During the early phase of this lytic cycle the rep-genes are expressed.
Approximately 12 to 16 hours later the capsid proteins VP1, VP2 and VP3 are produced and the replicated virus DNA is packaged into virions (structure of the AAV-genome and its genes is depicted in FIG. 2). The virions accumulate in the nucleus of the cell and are released when the cell lyses as a result of the accumulation of AAV and the helpervirus (reviewed in (Berns, 1990a; Berns, 1990b)).
The AAV-genome contains two genes rep and cap (FIG. 2). Three promoters (P5, P19 and P40) drive the synthesis of mRNAs coding for 4 Rep-proteins (Rep78, Rep68, Rep52 and Rep40) and three capsid proteins (VP1, VP2 and VP3). The AAV-genome is flanked on both sides by a 145 bp sequence, called the Inverted Terminal Repeat (TR), which appears to contain all the cis-acting sequences required for virus integration, replication and encapsidation (Lusby et al., 1980; Samulski et al., 1989).
The capsid proteins VP1, VP2 and VP3 are produced from a 2.6 kb transcript of the AAV P40 promoter, which is spliced into two 2.3 kb mRNAs by using the same splice donor but two different splice acceptor sites. The splice acceptor sites are located at both sides of the VP1 translation start signal. VP1 is translated from the messenger that uses the splice acceptor directly in front of the VP1 translation initiation codon. VP2 and VP3 are translated from messengers that are spliced to the acceptor 3xe2x80x2 of the VP1 ATG. VP2 and VP3 are translated from this messenger by use of an ACG translation start (VP2) or a downstream ATG (VP3). Since all three coding regions are in frame, the capsid proteins share a large domain with an identical amino-acid sequence. VP3 is entirely contained within VP1 and VP2, but the latter two contain additional amino-terminal sequences. Similarly, VP1 contains the entire VP2 protein but carries an additional N-terminal sequence. All three capsid proteins terminate at the same position (Ruffing et al., 1994). The AAV capsid is 20 to 24 nm in diameter (Berns and Bohensky, 1987; Srivastava et al., 1983) and contains approximately 5% VP1, 5% VP2 and 90% VP3. This ratio is believed to reflect the relative abundance of the alternatively spliced messengers and the reduced translation initiation efficiency at the ACG initiation codon for VP2.
During a productive infection, the P5-promoter is activated first and directs the production of the large Rep-proteins, Rep78 and Rep68. These proteins are essential for AAV-replication and in trans regulate the expression of viral and cellular genes. The large Rep-proteins activate the P19 and the P40 promoter. In a latent infection, however, Rep78 and Rep68 down regulate expression of the P5 promoter and help to maintain the latency of AAV (for a review see (Berns, 1990b)). The smaller Rep-proteins, Rep52 and Rep40 are encoded by transcripts from the P19 promoter and are important for the formation of infectious virus (Chejanovsky and Carter, 1989). The P40 promoter is the last promoter to become activated and its activation follows the expression of the late genes of the helper adenovirus. Via alternative splicing different mRNA are produced coding for the structural proteins VP1, VP2 and VP3 (Trempe and Carter, 1988).